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Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones

Collins, Cynthia H. and Arnold, Frances H. and Leadbetter, Jared R. (2005) Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. Molecular Microbiology, 55 (3). pp. 712-723. ISSN 0950-382X. http://resolver.caltech.edu/CaltechAUTHORS:COLmm05

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Abstract

LuxR-type transcriptional regulators play key roles in quorum-sensing systems that employ acyl-homoserine lactones (acyl-HSLs) as signal molecules. These proteins mediate quorum control by changing their interactions with RNA polymerase and DNA in response to binding their cognate acyl-HSL. The evolutionarily related LuxR-type proteins exhibit considerable diversity in primary sequence and in their response to acyl-HSLs having acyl groups of differing length and composition. Little is known about which residues determine acyl-HSL specificity, and less about the evolutionary time scales required to forge new ones. To begin to examine such issues, we have focused on the LuxR protein from Vibrio fischeri, which activates gene transcription in response to binding its cognate quorum signal, 3-oxohexanoyl-homoserine lactone (3OC6HSL). Libraries of luxR mutants were screened for variants exhibiting increased gene activation in response to octanoyl-HSL (C8HSL), with which wild-type LuxR interacts only weakly. Eight LuxR variants were identified that showed a 100-fold increase in sensitivity to C8HSL; these variants also displayed increased sensitivities to pentanoyl-HSL and tetradecanoyl-HSL, while maintaining a wild-type or greater response to 3OC6HSL. The most sensitive variants activated gene transcription as strongly with C8HSL as the wild type did with 3OC6HSL. With one exception, the amino acid residues involved were restricted to the N-terminal, 'signal-binding' domain of LuxR. These residue positions differed from critical positions previously identified via 'loss-of-function' mutagenesis. We have demonstrated that acyl-HSL-dependent quorum-sensing systems can evolve rapidly to respond to new acyl-HSLs, suggesting that there may be an evolutionary advantage to maintaining such plasticity.


Item Type:Article
Additional Information:© 2004 Blackwell Publishing Ltd. Accepted 13 October, 2004. We are grateful to Bernard Hauer for providing synthetic C5HSL. We thank M. Barnet, T. Ciche, J. Gralnick, J. Silberg, M. Urbanowski and L. You for critical reading of the manuscript. This research was supported by the Defense Advanced Research Projects Agency (DARPA) grant N66001-02-8929 and by National Science Foundation, Biological Information Technology and Storage (BITS) grant EIA-0130613. Disclaimer: Any opinions, findings and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the DARPA.
Subject Keywords:SENSING TRANSCRIPTION FACTOR; TO-CELL COMMUNICATION; PSEUDOMONAS-AERUGINOSA; LUMINESCENCE GENES; METABOLIC PATHWAYS; BINDING REGION; AUTOINDUCER; PROTEIN; SPECIFICITY; SYSTEM
Record Number:CaltechAUTHORS:COLmm05
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:COLmm05
Alternative URL:http://dx.doi.org/10.1111/j.1365-2958.2004.04437.x
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:5553
Collection:CaltechAUTHORS
Deposited By: Lindsay Cleary
Deposited On:25 Oct 2006
Last Modified:26 Dec 2012 09:13

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